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Molecular Pathology

Polymerase Chain Reaction

Real-time PCR

 

Author: Rodney E. Shackelford, D.O., Ph.D. (see Reviewers page)

Revised: 22 September 2012, last major update July 2010

Copyright: (c) 2008-2010, PathologyOutlines.com, Inc.

 

General

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Also called quantitative real-time PCR, this technique simultaneously amplifies and quantifies the amount of a specific DNA sequence during amplification by comparing the sample being amplified to standardized controls, hence the term “real-time”

The quantification may be the relative amount of DNA compared to a known standard or amplification copy number

● Initially, agents such as ethidium bromide were added to PCR reactions to quantify DNA during amplification

● Ethidium bromide can detect increasing DNA concentrations in closed-tube PCR reactions following excitation at 254 nm UV light

● However ethidium bromide has several drawbacks, including (a) it is a powerful mutagen; (b) its fluorescence is not specific for double-stranded DNA; and (c) it can inhibit DNA polymerase activity

 

SYBR green

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● In contrast to ethidium bromide, is specific for double-stranded DNA or for the amplified DNA sequence:

● A cyanine dye which specifically binds double-stranded DNA, with a double-stranded DNA bound fluorescence quantum yield over 5x greater than the DNA/ethidium bromide complex; absorbs at 488 nm (blue) and strongly emits at 522 nm (green)

To quantify DNA amplification with SYBR Green, the dye is added to the PCR reaction and the relative sample fluorescence is measured, and compared to standard controls upon completion of each thermocycle (i.e., when double-stranded DNA has formed with the reaction tube)

Since the concentration of double-stranded DNA is measured, only the relative amount of DNA can be determined, not the exact amplification number

While this technique works well, it has the drawback that SYBR Green binds to all double-stranded DNA equally; thus any nonspecific double-stranded DNA in the reaction, for example “primer dimers” is also measured

 

Probe based detection systems

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This technique lessens problems associated with measuring nonspecific double-stranded DNA in PCR reactions

A DNA or RNA probe specific for a sequence within the DNA to be amplified and inside the oligonucleotide binding sites is made which carries a closely apposed fluorescent reporter and fluorescence quencher

When placed close to the reporter, the quencher prevents fluorescent detection

The PCR reaction is run with the standard reagents and the labeled probe

Following primer and probe annealing, the 5' to 3' exonuclease Taq polymerase activity breaks down the probe during the elongation phase

As a result, the reporter and quencher are no longer in close proximity and the reporter fluorescence can be detected

The amount or reporter fluorescent activity is proportional to the amount of specific target DNA synthesized

The amount of specific transcript can be read anytime within each thermocycle, as reporter activity is not altered by the present of absence of double-stranded DNA

Since early PCR thermocycles will initially produce only tiny amounts of free reporter, early RT-PCR thermocycles are characterized by little or no increase in fluorescent signal detection

Later thermocycles will reach a point where fluorescence levels are above non-specific background fluorescence and increasing fluorescence can be plotted against thermocycle number on a logarithmic scale

The point at which the fluorescent signal exceeds the background level (i.e., threshold) is the cycle threshold number or Ct value

The Ct is inversely proportional to the amount of target DNA; the lower the Ct level, the greater the initial concentration of DNA

Comparison of the Ct vs. thermocycle number allows the quantification of the DNA / RNA concentration in the original sample

In general, a Ct below 29 shows abundant nucleic acid in the original sample, 30-37 shows moderate amounts and 38-40 shows very low amounts of target nucleic acid and may even represent contamination of the original sample

Since PCR doubles the amount of target nucleic acid with every thermocycle, each thermocycle one unit increase between two nucleic acid samples represents 50% less nucleic acid in the sample with the higher Ct value

Often the amount of nucleic acid is determined by plotting the Ct value of a sample to a standard curve of an amplifiable nucleic acid

 

End of Molecular Pathology > Polymerase Chain Reaction > Real-time PCR

 

 

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